The long-term objectives of this application are to elucidate the mechanisms and regulation of renal tubular calcium transport. The specific aims of this proposal are to investigate the plasmalemmal transport mechanisms regulating cytosolic free calcium concentration and epithelial calcium absorption in cells of the rabbit connecting tubule and cortical thick ascending limb of Henle's loop, which are important calcium-reabsorbing segments of the nephron. Rabbits will be sacrificed and individual connecting tubules and cortical thick ascending limbs will be dissected from their kidneys and perfused in vitro. Cytosolic free concentrations of calcium, sodium, and hydrogen ions will be measured in segments (photon-counting photometry) and individual cells (photon-counting photometry and digital video - imaging) by quantitative fluorescence microscopy. The compositions of both luminal perfusion and peritubular bathing solutions will be manipulated independently and in concert to identify plasmalemmal calcium transport processes at both the luminal and basolateral sides of the cell. In addition to the roles of sodium and hydrogen ions, the actions of parathyroid hormone, the main calcium-regulating hormone in the kidney, will be studied. The regulation of urinary calcium excretion is important to calcium homeostasis. Hypercalciuria, a major determinant of renal stone disease, is the primary disorder of urinary calcium excretion in humans. The proposed studies will provide new information on the cellular and plasmalemmal handling of calcium within the nephron that will advance our knowledge in this area from the epithelial to the cell membrane level.